The proposal for continuation of this Program Project Grant (PPG) is inspired by new opportunities to understand mechanisms of neurotransmitter transporters (NTs) in the physiological processes underlying neurotransmission in general, and their involvement in drug addiction and abuse in particular. The focus is on the transporter systems for dopamine (DAT), serotonin (SERT), and norepinephrine (NET), with generalizations and comparisons to other Na+Cl- dependent biogenic amine transporters. The broad aims are to achieve a mechanistic characterization, in a detailed structural context, of multifaceted NT function and modulation by substrates and ligands. The aims address regulation and modulation of NT functional characteristics by interactions with their environment (e.g., by oligomerization, scaffold-binding, etc.) and by phosphorylation of the intracellular segments (N- and C-terminals, and loops). The studies aim to discover functional and structural properties of interactions 1) among specific NT structural elements, and 2) with other proteins such as PDZ domains and kinases, in key processes including cell surface expression and stabilization, and the regulation of substrate uptake and efflux. Such insights are essential to understand mechanisms responsible for pharmacological properties of abused substances and stimulants (e.g., cocaine, amphetamine and MDMA-ecstasy) and of major classes of therapeutic agents (e.g., antidepressants) that act directly on NTs. The synergistic construct of the PPG makes it possible to address the regulation of NT function with multidisciplinary approaches in collaborations and coordinated experiments using electrophysiological and pharmacological assays of NT activity, as well as biochemical procedures (such as cysteine cross-linking and accessibility scans) in wild type, mutant, phosphorylated and fused-protein expression constructs (in Projects 2-4 and Core B), biophysical measurements and computational evaluations of structure, affinity and selectivity in protein-protein interactions (in Projects 2 and 1), discovery (in Project 2) and interpretation of selectivity in high-throughput protein-interaction microarrays (in Projects 2 and 1), and shared contexts of structural models and data management systems provided by Project 1 and Core A.